YKL-40 was first identified as a protein secreted in large amounts by a human osteosarcoma cell line MG63 in vitro and was named according to the last three N-terminal amino acids of its amino acid sequence, Tyr (Y), Lys (K) and Leu (L), and approximate molecular weight of 40 kDa (Johansen et al., 1992).
Analysis of the amino acid sequence of YKL-40 revealed that the protein belongs to the glycosyl hydrolase family 18 (Hakala et al., 1993). The family consists of enzymes including chitinases and also proteins, which do not have enzymatic activity. The gene for YKL-40 (CHI3L1) is located on chromosome 1q31-1q32 and consists of 10 exons and spans about 8 kilobases of genomic DNA (Rehli et al., 1997). The Sp1-family transcription factors seem to have a predominantly role in controlling YKL-40 promoter activity (Rehi et al., 2003). The crystallographic structure of human YKL-40 has been described (Fusetti et al., 2003; Houston et al., 2003). The protein contains two globular domains: a big core domain which consists of a (β/α)8 domain structure with a triose-phosphate isomerase (TIM) barrel fold, and a small α/β domain, composed of five antiparallel β-strands and one α-helix, inserted in the loop between strand β7 and helix α7. This confers to the active site of YKL-40 a groove-like character. The structure of YKL-40 is in part similar to the structure of several proteins, e.g. human chitotriosidase (Fusetti et al., 2002), mouse Ym1 (Sun et al., 2001), Drosophila melanogaster imaginal disc growth factor-2 (Varela et al., 2002) and some other members of the glycosyl hydrolase family 18 (Coulson, 1994), but there are also major differences. One of these differences is a mutation of one of the three amino acids (Asp, Glu and Asp) essential for the chitinase-like catalytic activity, namely Glu→Leu, which completely rules out the function of YKL-40 as a glycolytic enzyme. YKL-40 can binds chitin, but has no enzymatic activity (Renkema et al., 1998). YKL-40 is a glycoprotein and this is also a unique feature of YKL-40 comparing to the other mammalian chitinase-like proteins.
YKL-40 can bind heparin. The amino acid sequence of the protein contains one heparin binding motif (143GRRDKQH149) which is located in a surface loop (Fusetti et al., 2003). YKL-40 can also bind hyaluronan. The folded protein contains two potential hyaluronan binding sites on the external face. Human YKL-40 can bind chitin of different lengths in a similar fashion as chitinases of the Family 18. Nine sugar-binding subsites were identified in the 43 Å groove of YKL-40 (Fusetti et al., 2003). Binding of short or long oligosaccharides to human YKL-40 is also possible. The presence of two distinct binding sites with selective affinity for long and short oligosaccharides (not found in other mammalian chitinase-like proteins) may have significance for the functional activity of YKL-40 as a cross-linker between two targets carrying these oligosaccharides.
YKL-40 possesses a number of biological activities. Thus, it has been shown that human YKL-40 can act as a growth factor for cells of connective tissue, such as chondrocytes and synovial cells (De Ceuninck et al., 2001; Recklies et al., 2002). YKL-40 also promotes the growth of fibroblasts in a fashion similar to insulin-like growth factor 1 (IGF-1) (Recklies et al., 2002). In fibroblasts YKL-40 initiates activation of MAP kinase and PI3K signalling cascades leading to phosphorylation of both the extracellular signal-regulated kinase (ERK)-1/2 MAP kinase and protein kinase B (AKT). Activation of cytoplasmic signal-transduction pathways by YKL-40 has been suggested to be mediated through interaction of YKL-40 with one or more receptor molecules on the plasma membrane, but the identity of the cellular receptor mediating the biological effects of YKL-40 is currently unknown.
It has also been demonstrated that YKL-40 acts as a chemoattractant for endothelial cells and stimulates migration of these cells comparable to stimulation by basic fibroblast growth factor (Malinda et al., 1999).
YKL-40 modulates vascular endothelial cell morphology by promoting the formation of branching tubules, indicating that YKL-40 may play a role in angiogenesis by stimulating the migration and reorganization of vascular endothelial cells (Malinda et al., 1999). YKL-40 is also an adhesion and migration factor for vascular smooth muscle cells (Nishikawa et al., 2003). It has been suggested that YKL-40 may affect the local extracellular hyaluronan concentrations available for cell attachment via two independent mechanisms: by binding to the extracellular hyaluronan, and by interfering with the synthesis and secretion of hyaluronan by cells. Thus, YKL-40 may influence the extent of cell adhesion and migration during the tissue remodeling processes that take place during inflammation, fibrosis, atherogenesis and cancer growth and cancer metastasis.
In mice YKL-40 was called the “breast regression protein (Brp-39)” because the expression of the protein was induced in mammary epithelial cells a few days after weaning. It was proposed (Mohanty et al., 2003) that YKL-40 is involved in regulation of programmed cell death during mammary involution as a protective signalling factor that determines which cells are to survive the drastic tissue remodelling that occurs during involution.
YKL-40 is expressed by different types of cells in vitro and in vivo, in particular in tissues characterised by inflammation, degradation/remodeling of the extracellular matrix or ongoing fibrogenesis. YKL-40 is secreted by activated neutrophils (Volck et al., 1998), by macrophages during late state of differentiation (Kirckpatrick et al., 1997; Krause et al. 1996; Rehli et al., 1997; Renkema et al., 1998; Rehli et al., 2003), arthritic chondrocytes (Hakala et al., 1993; Johansen et al. 2001; Volck et al. 2001), differentiated vascular smooth muscle cells (Shackelton et al., 1995; Malinda et al., 1999; Nishikawa et al. 2003) and fibroblast-like synovial cells (Hakala et al. 1993; Nyirkos et al., 1990; Dasuri et al., 2004). Studies in human fetal chondrocytes indicate that YKL-40 is a differentiation marker (Imabayashi et al., 2003). In vivo YKL-40 mRNA and proteins expression are found by a subpopulation of macrophages in inflamed synovial membrane (Kirkpatrick et al., 1997; Baeten et al., 2000; Volck et al., 2001), atheromatous plaques (Boot et al. 1999), arteritic vessels from patients with giant cell arteritis (Johansen et al., 1999a) and by arthritic chondrocytes (Volck et al., 2001), and peritumoral macrophages in biopsies from small cell lung cancer express YKL-40 mRNA (Junker et al., 2005b).
A strong expression of YKL-40 mRNA in human liver has been shown to be associated with the presence of fibrosis. Immunohistochemical studies of liver biopsies have shown YKL-40 protein expression in areas of the liver with fibrosis, whereas no expression was observed in hepatocytes (Johansen et al., 1997; Johansen et al. 2000). Suppression subtractive hybridization analysis and RT-PCR have demonstrated that YKL-40 is one of the most overexpressed proteins in cirrhotic liver tissue caused by hepatitis C virus (HCV) (Shackel et al., 2003).
Patients with non-malignant diseases characterized by inflammation and fibrosis such as active rheumatoid arthritis (Johansen et al., 1993; Harvey et al., 1998; Johansen et al., 1999b; Volck et al., 2001), severe bacterial infections (Nordenbaek et al., 1999; Kronborg et al., 2002), active inflammatory bowel disease (Koutroubakis et al., 2003; Vind et al., 2003), and liver fibrosis (Johansen et al., 1997; Johansen et al., 2000; Tran et al., 2000; Nøjgaard et al., 2003) have elevated serum YKL-40.
YKL-40 is expressed and secreted by several types of human carcinoma (breast, colon, lung, kidney, ovarian, prostate, uterine, osteosarcoma, oligodendroglioma, glioblastoma and germ cell tumors) (A search of the YKL-40 sequence against the dbest database at the National Center for Biotechnology Information; Johansen et al., 1992; Junker et al., 2005a), and by murine mammary tumors initiated by neu/ras oncogenes (Morrison et al., 1994). Microarray gene analyses have identified the YKL-40 gene as one of the most overexpressed genes in papillary thyroid carcinoma (Huang et al., 2001), high-grade malignant gliomas (Tanwar et al., 2002), and extracellular myxoid chondrosarcoma (Sjogren et al., 2003). YKL-40 is expressed and secreted in vitro by the osteosarcoma cell line MG63, glioblastoma cells and myeloid leukemia cell lines (U937, THP-1, HL-60) (Johansen et al., 1992; Rehli et al., 2003; Kirkpatrick et al., 1995; Verhoeckx et al., 2004). YKL-40 is not expressed by small cell lung cancer cell lines in vitro nor in vivo but strongly expressed by tumor associated macrophages in small cell lung cancer biopsies (Junker et al., 2005b).
A number of studies has now reported an elevated level of YKL-40 protein in serum of cancer patients (Johansen et al., 1995; Cintin et al., 1999; Cintin et al., 2002; Tanwar et al., 2002; Brasso et al., 2003; Dehn et al., 2003; Geertsen et al., 2003; Høgdall et al., 2003; Jensen et al., 2003; Johansen et al., 2003; Dupont et al., 2004; Johansen et al., 2004). Several studies have demonstrated that an elevated serum concentration of YKL-40 in patients with breast-, colorectal-, ovarian-, kidney-, small cell lung-, and prostate carcinomas is an independent prognostic parameter of short recurrence free interval and short overall survival. This observation has been done in patients with local or advanced cancer at time of first cancer diagnosis and at time of relapse (Johansen et al., 1995; Cintin et al., 1999; Cintin et al., 2002; Brasso et al., 2003; Dehn et al., 2003; Geertsen et al., 2003; Høgdall et al., 2003; Jensen et al., 2003; Johansen et al., 2003; Dupont et al., 2004; Johansen et al., 2004). Based on these and other findings YKL-40 was suggested as a diagnostic marker of the presence or absence of a cancer and for the prognosis of cancer recurrence and survival of cancer patients (WO 00/19206), and it was described as a marker for degradation of connective tissue and used in methods for identifying the presence of a disease associated with degradation of connective tissue (e.g. cancer) described (WO 95/01995; U.S. Pat. No. 5,935,798). Both groups of latter methods are based on employing an anti-YKL-40 antibody for detecting the protein in samples from the patients.
Antibodies against YKL-40 has long been known in the art and used for example for the detection and monitoring the level of YKL-40 in blood serum/plasma of cancer patients, however, functional anti-YKL-40 antibodies, which would be capable of inhibiting the function of YKL-40, have not been produced nor described.